Low Concentrations Of Caffeine Research Articles

Immunomodulatory properties of caffeine and caffeine-treated immune cells in depression-like state

Depression is one of the leading global health problems worldwide. A significant increase in prevalence among the working-age population, as well as high comorbidity, partial or complete drug resistance in a third of patients determines the need to develop new approaches to the treatment of depression. Violation of mutual regulation of the main homeostatic systems plays an important role in the pathogenesis of depression. Psycho- and immunopathology are closely interrelated: pathological changes in the functioning of both systems occur simultaneously and are interdependent. This determines the prospects for the treatment of depression based on immunological approaches. Caffeine, a drug known for its psychoneuromodulatory properties, is an adenosine receptor antagonist with a pronounced dose-dependent effect. Adenosine receptors are expressed by both CNS cells and cells of the immune system, which determines its immunomodulatory properties. The similarity of both phenotypes and functions of the cellular elements of the immune and nervous systems, as well as the unidirectional effect of most psychoactive drugs on the central nervous system and the immune system, determines the interest in studying the immunomodulatory properties of caffeine for a targeted effect on the functional activity of immune cells, with a view to their subsequent use as model objects for the normalization of neuroimmune regulatory connections disturbed in a depressive state. Previously, we first demonstrated the possibility of editing depression-like behavior by immune cells precultivated with caffeine and showed the central mechanisms of this effect aimed at stimulating neuroplasticity processes and reducing neuroinflammation. The aim of this study was to evaluate the functional phenotype of immune cells in depressive-like animals after in vitro treatment of cells with caffeine, as well as the effects of transplantation of caffeine-precultured immune cells on the parameters of the functional activity of the immune system of syngeneic depressive-like recipients. As a result of the study, it was shown that low concentrations of caffeine increase the spontaneous and mitogen-induced proliferative activity of splenocytes of depression-like male mice (CBA x C57BL/6)F1 in vitro ; this changes the spontaneous and mitogen-stimulated production of cytokines TNFa IL-1b, IFNg, IL-2, and IL-10 by these cells. After intravenous administration of the precultured with caffeine depression-like donor’s splenocytes to syngeneic depression-like recipients, stimulation of the humoral immune response was observed in the latter, assessed by an increase in both the relative and absolute number of antibody-forming spleen cells. Stimulation of spontaneous proliferative activity of lymphocytes in splenocyte culture was also registered. The data obtained indicate a positive effect of caffeine in vitro on the immune cell’s functional activity, as well as a positive immunomodulatory effect of the immune cells precultured with caffeine in a depression-like state in vivo.

Caffeine Consumption Helps Honey Bees Fight a Bacterial Pathogen.

Caffeine has long been used as a stimulant by humans. Although this secondary metabolite is produced by some plants as a mechanism of defense against herbivores, beneficial or detrimental effects of such consumption are usually associated with dose. The Western honey bee, Apis mellifera, can also be exposed to caffeine when foraging at Coffea and Citrus plants, and low doses as are found in the nectar of these plants seem to boost memory learning and ameliorate parasite infection in bees. In this study, we investigated the effects of caffeine consumption on the gut microbiota of honey bees and on susceptibility to bacterial infection. We performed in vivo experiments in which honey bees, deprived of or colonized with their native microbiota, were exposed to nectar-relevant concentrations of caffeine for a week, then challenged with the bacterial pathogen Serratia marcescens. We found that caffeine consumption did not impact the gut microbiota or survival rates of honey bees. Moreover, microbiota-colonized bees exposed to caffeine were more resistant to infection and exhibited increased survival rates compared to microbiota-colonized or microbiota-deprived bees only exposed to the pathogen. Our findings point to an additional benefit of caffeine consumption in honey bee health by protecting against bacterial infections. IMPORTANCE The consumption of caffeine is a remarkable feature of the human diet. Common drinks, such as coffee and tea, contain caffeine as a stimulant. Interestingly, honey bees also seem to like caffeine. They are usually attracted to the low concentrations of caffeine found in nectar and pollen of Coffea plants, and consumption improves learning and memory retention, as well as protects against viruses and fungal parasites. In this study, we expanded these findings by demonstrating that caffeine can improve survival rates of honey bees infected with Serratia marcescens, a bacterial pathogen known to cause sepsis in animals. However, this beneficial effect was only observed when bees were colonized with their native gut microbiota, and caffeine seemed not to directly affect the gut microbiota or survival rates of bees. Our findings suggest a potential synergism between caffeine and gut microbial communities in protection against bacterial pathogens.

NOVEL UV SPECTROSCOPIC METHOD FOR QUANTIFICATION OF CAFFEINE IN MARKETED ENERGY DRINKS

Objective: This study is performed to quantitatively estimate caffeine in marketed energy drinks by using UV-Visible spectroscopic method.
 Methods: This experiment was performed on various soft drinks and energy drinks available in the local market of India to determine the caffeine concentration. The quantitative method used was simple, easy UV-Visible spectrophotometric method by using carbon dichloromethane as diluent at 274 nm. UV-Vis spectroscopy is an analytical technique that measures the amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted through a sample in comparison to a reference or blank sample.
 Results: Among all the samples i.e. soft or energy drinks taken for this experiment sample 1 has low concentration of caffeine and the highest concentration was observed in sample 3.
 Conclusion: Caffeine in an energy drink provides a stimulant effect, it gives energy. At lower levels, as it’s typically used in soft drinks, it has less of a stimulant effect and is used mainly for its taste profile. However the concentration of caffeine should be within the limits specified. Excessive consumption of caffeine may lead to anxiety, caffeine dependence, increased urination, and may cause insomnia. Energy drinks can contain high levels of caffeine but are unlikely to be hazardous unless consumed with alcohol. This research is very important analytical process to safeguard the well being of people who are unaware to adverse effects of caffeine.

Correlation between caffeine and coprostanol in contrasting Amazonian water bodies

The evaluation of contamination by domestic sewage is relevant in the Amazon region; however, it has neither been well-developed nor accompanied by research or monitoring programs. In this study, caffeine and coprostanol as indicators of sewage were investigated in water samples from Amazonian water bodies that crisscross the city of Manaus (Amazonas state, Brazil) and cover regions with distinct main land uses such as high-density residential, low-density residential, commercial, industrial, and environmental protection areas. Thirty-one water samples were studied based on their dissolved and particulate organic matter (DOM and POM) fractions. Quantitative determination of both caffeine and coprostanol was carried out using LC-MS/MS with APCI in the positive ionization mode. The streams of the urban area of Manaus had the highest concentrations of caffeine (1.47–69.65 μg L−1) and coprostanol (2.88–46.92 μg L−1). Samples from the peri-urban Tarumã-Açu stream and from the streams in the Adolpho Ducke Forest Reserve showed much lower concentrations of caffeine (20.20–165.78 ng L−1) and coprostanol (31.49–120.44 ng L−1). Samples from the Negro River showed a wider range of concentrations of caffeine (20.59–873.59 ng L−1) and coprostanol (31.72–706.46 ng L−1), with the highest values found in the outfalls of the urban streams. Levels of caffeine and coprostanol were significantly positively correlated in the different organic matter fractions. The coprostanol/(coprostanol + cholestanol) ratio proved to be a more suitable parameter than the coprostanol/cholesterol one in low-density residential areas. Proximity to densely populated areas and the flow of water bodies appear to influence the caffeine and coprostanol concentrations, which was observed in their clustering in the multivariate analysis. The results indicate that caffeine and coprostanol can be detected even in water bodies that receive very low domestic sewage input. Therefore, this study revealed that both caffeine in DOM and coprostanol in POM represent viable alternatives for use in studies and monitoring programs even in remote areas of the Amazon, where microbiological analyses are often unfeasible.

Effect of low concentrations of caffeine and colchicine on microbial growth and biofilm formation

A study into the effect of low concentrations of caffeine and colchicine alkaloids on the growth and biofilm formation of rhizospheric microorganisms – Gram-positive Rhodococcus qingshengii and Gramnegative Rhizobium radiobacter – is presented. Alkaloids present in plants effectively protect them from being eaten by animals of various taxa, as well as from fungal and bacterial infections. Forming part of a complex, evolutionary system of plant-microbial interactions, they occur naturally in the medium at low concentrations. One of the decisive factors in the spread of microorganisms is the formation of biofilms. In the study, alkaloids were added to the bacterial suspension at time intervals corresponding to distinct phases of biofilm formation. In order to determine the level of biofilm formation, bacterial cells were stained with crystal violet. The optical density of the suspension was used to assess the effect of alkaloids on bacterial growth and biofilm formation. The effect of caffeine and colchicine in concentrations of 10-5 g/L on the growth and biofilm formation of microorganisms is revealed for the first time. Although alkaloids did not affect the growth of the suspension of Gram-positive Rhodococcus qingshengii, they effectively suppressed the suspension of Gram-negative Rhizobium radiobacter. While Rhodococcus qingshengii showed greater sensitivity to alkaloids at the stage of biofilm formation, Rhizobium radiobacter was more sensitive at the stage of "mature" biofilms. The effect of alkaloids could be expressed both in terms of suppression and enhancement of biofilm growth. It can be assumed that the mechanism of action of plant alkaloids at the level of signaling systems and bacterial quorum sensing allows the protective function to be maintained even with a significant decrease in the number of alkaloids under conditions of permanent antagonism in which they are constantly being destroyed by phytopathogens.

Investigation of potential behavioral and physiological effects of caffeine on D. magna.

With the ever-increasing consumption of pharmaceutical compounds, their presence in the environment is now an undisputable reality. The majority of these compounds are released into the wild after their therapeutic use, as biotransformation products or in their original form. The presence of this class of compounds in the environment, due to their biological properties, can exert effects on non-target organisms, with adverse consequences. In addition, some bioactive substances, such as stimulants of the central nervous system, are also used by humans as part of their diet. The adverse consequences posed by such chemicals may be permanent or transient, if the exposure to xenobiotics is halted; it is thus of the paramount importance to study effects that result from long-term exposure to toxicants, but also the recovery of organisms previously exposed to such substances, especially if such chemicals may cause some type of addiction. Caffeine (1,3,7-trimethylxanthine) is a naturally occurring alkaloid found in many plants, being one of the most common stimulant/pharmaceutical compounds found in the environment. In addition, it is addictive, and strongly consumed by humans, a factor that contributes also for its continuous presence in the aquatic environment. The aim of this study was to evaluate the effects of environmentally relevant concentrations (0.08; 0.4; 2; 10; and 50μg/L) of caffeine on behavior and physiological parameters (that are proxies of metabolic traits, such as oxygen uptake and glycogen content), in individuals of the freshwater crustacean species Daphnia magna, of distinct ages, and with or without a recovery period in the absence of caffeine. Regarding behavior, the results indicated that caffeine exposure altered the moved distance of the test organisms, but not according to a coherent pattern; low concentrations of caffeine reduced the movement of exposed daphnids, while higher levels did not have any measurable effect on this parameter. In addition, it was possible to identify subtle withdrawal effects (animals exposed to caffeine during 21days and kept in uncontaminated media for 2days). Regarding the other two studied parameters, caffeine exposure did not result in any significant modification in oxygen uptake and glycogen stores/reserves of the test organisms, in animals continuously exposed, or in those subjected to a recovery period, suggesting that despite a behavioral stimulatory effect, this was not followed by any metabolic change, and no addictive effect was possible to infer. The results showed that the presence of caffeine in environmental concentrations can induce mild behavioral effects at low, albeit realistic levels, but not capable of establishing clear biochemical changes.

Binding Modes and Metabolism of Caffeine.

A correct estimate of ligand binding modes and a ratio of their occupancies is crucial for calculations of binding free energies. The newly developed method BLUES combines molecular dynamics with nonequilibrium candidate Monte Carlo. Nonequilibrium candidate Monte Carlo generates a plethora of possible binding modes and molecular dynamics enables the system to relax. We used BLUES to investigate binding modes of caffeine in the active site of its metabolizing enzyme Cytochrome P450 1A2 with the aim of elucidating metabolite-formation profiles at different concentrations. Because the activation energies of all sites of metabolism do not show a clear preference for one metabolite over the others, the orientations in the active site must play a key role. In simulations with caffeine located in a spacious pocket above the I-helix, it points N3 and N1 to the heme iron, whereas in simulations where caffeine is in close proximity to the heme N7 and C8 are preferably oriented toward the heme iron. We propose a mechanism where at low caffeine concentrations caffeine binds to the upper part of the active site, leading to formation of the main metabolite paraxanthine. On the other hand, at high concentrations two molecules are located in the active site, forcing one molecule into close proximity to the heme and yielding metabolites theophylline and trimethyluretic acid. Our results offer an explanation of previously published experimental results.

Caffeine Content and Related Gene Expression: Novel Insight into Caffeine Metabolism in Camellia Plants Containing Low, Normal, and High Caffeine Concentrations.

Caffeine is a crucial secondary metabolic product in tea plants. Although the presence of caffeine in tea plants has been identified, the molecular mechanisms regulating relevant caffeine metabolism remain unclear. For the elucidation of the caffeine biosynthesis and catabolism in Camellia plants, fresh, germinated leaves from four Camellia plants with low (2), normal (1), and high (1) caffeine concentrations, namely, low-caffeine tea 1 (LCT1, Camellia crassicolumna), low-caffeine tea 2 (LCT2, C. crassicolumna), Shuchazao (SCZ, C. sinensis), and Yunkang 43 (YK43, C. sinensis) were used in this research. Transcriptome and purine alkaloids analyses of these Camellia leaves were performed using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Moreover, 15N-caffeine tracing was performed to determine the metabolic fate of caffeine in leaves of these plants. Caffeine content was correlated with related gene expression levels, and a quantitative real-time (qRT) PCR analysis of specific genes showed a consistent tendency with the obtained transcriptomic analysis. On the basis of the results of stable isotope-labeled tracer experiments, we discovered a degradation pathway of caffeine to theobromine. These findings could assist researchers in understanding the caffeine-related mechanisms in Camellia plants containing low, normal, and high caffeine content and be applied to caffeine regulation and breeding improvement in future research.

Pro-oxidant and lifespan extension effects of caffeine and related methylxanthines in Caenorhabditis elegans

Caffeine and related purine alkaloids are common ingredients of many stimulating drinks. Studies have shown that lower concentrations of caffeine have a protective role in aging-related disorders. However, the associated mode of action of caffeine and its related methylxanthines is still not clear. In this study, we demonstrated that caffeine and theophylline promote longevity in Caenorhabditis elegans. Lifespan studies with the wild type, DAF-16 and SKN-1 mutant strains indicated that the methylxanthines-mediated lifespan extension in C. elegans was independent of DAF-16/FOXO and SKN-1. All the tested methylxanthines could protect C. elegans against acute oxidative stress. At early stages of life, an increase of ROS (reactive oxygen species) induced the translocation of DAF-16 and SKN-1, resulting in upregulation of several antioxidant genes, for example, sod-3p::GFP, gst-4p::GFP, gcs-1p::GFP; and downregulation of hsp-16.2p::GFP. RT-PCR corroborates the upregulation of gst-4 and skn-1 genes. The expression of DAF-16 decreased although its nuclear translocation was induced.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca2+ signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca2+ channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca2+ levels. Caffeine sensitizes RyR to Ca2+ and promotes ER Ca2+ release at basal cytosolic Ca2+ levels. This outcome is frequently used as a readout for the presence of CICR. By monitoring ER luminal Ca2+ with the low-affinity genetic Ca2+ probe erGAP3, we find here that application of 50 mM caffeine rapidly reduces the Ca2+ content of the ER in HeLa cells by ∼50%. Interestingly, this apparent ER Ca2+ release does not go along with the expected cytosolic Ca2+ increase. These results can be explained by Ca2+ chelation by caffeine inside the ER. Ca2+-overloaded mitochondria also display a drop of the matrix Ca2+ concentration upon caffeine addition. In contrast, in the cytosol, with a low free Ca2+ concentration (10-7 M), no chelation is observed. Expression of RyR3 sensitizes the responses to caffeine with effects both in the ER (increase in Ca2+ release) and in the cytosol (increase in Ca2+ peak) at low caffeine concentrations (0.3-1 mM) that have no effects in control cells. Our results illustrate the fact that simultaneous monitoring of both cytosolic- and ER Ca2+ are necessary to understand the action of caffeine and raise concerns against the use of high concentrations of caffeine as a readout of the presence of CICR.

Low Concentrations of Caffeine and Its Analogs Extend the Lifespan of Caenorhabditis elegans by Modulating IGF-1-Like Pathway.

Caffeine has been reported to delay aging and protect aging-associated disorders in Caenorhabditis elegans. However, the effects of low concentration of caffeine and its analogs on lifespan are currently missing. Herein, we report that at much lower concentrations (as low as 10 μg/ml), caffeine extended the lifespan of C. elegans without affecting food intake and reproduction. The effect of caffeine was dependent on IGF-1-like pathway, although the insulin receptor homolog, daf-2 allele, e1371, was dispensable. Four caffeine analogs, 1-methylxanthine, 7-methylxanthine, 1,3-dimethylxanthine, and 1,7-dimethylxanthine, also extended lifespan, whereas 3-methylxanthine and 3,7-dimethylxanthine did not exhibit lifespan-extending activity.

From the field to coffee cup: impact of planting design on chlorogenic acid isomers and other compounds in coffee beans and sensory attributes of coffee beverage

Various chemical compounds in green coffee beans are precursors of coffee beverage sensory attributes. The hypothesis was that those chemical compounds would vary with microclimate over the vertical plant profile of coffee plants. The plants of the cultivar IAPAR 59 were grown in two high plant densities (6000 and 10,000 plants ha−1) under rectangular and square planting patterns. Coffee beans were collected from three canopy strata. Chemical compounds and chlorogenic acid isomer (CGA) contents were determined by near infrared spectroscopy and ultra-performance liquid chromatography, respectively. The free-choice profile technique was employed to estimate the sensory attributes of coffee beverage. Brightness, transparency appearance, typical coffee color and aroma, tastes as sweet, acid, astringent and bitter, and full-bodied texture were attributes detected by panelists. Plants cultivated under lower plant density had lower leaf area index indicating lower self-shading than under higher plant density. Coffee beans originated from the inferior stratum of higher density had the lowest concentrations of 5-caffeoylquinic acid (5-CQA), while the inverse situation occurred in their upper strata. Strata of lower planting density were associated to high levels of all CGA isomers. The coffees cultivated under higher density showed high lipids and CGA isomers contents. Under lower density, beans had low concentration of sucrose, proteins, and caffeine and high of CGA isomer contents. The beverage sensory attributes depended on the concentration of proteins, lipids, and sucrose in combination to CQA isomers in beans, varying over the vertical profile and planting designs of coffee plants.

Caffeine Inhibits Fluid Secretion by Interlobular Ducts From Guinea Pig Pancreas.

Caffeine is contained in coffee, tea, and numerous beverages and foods. We examined the direct effects of caffeine on the physiological function of pancreatic duct cells by using interlobular duct segments isolated from guinea pig pancreas. The rate of fluid secretion was continuously measured by monitoring the luminal volume of isolated duct segments. Changes in intracellular Ca concentration ([Ca]i) were estimated by microfluorometry in ducts loaded with Fura-2. Both secretin-stimulated and acetylcholine (ACh)-stimulated fluid secretions were substantially and reversibly inhibited by relatively low concentrations of caffeine as low as 0.03 mM relevant to blood levels after ingestion of caffeine-containing beverages. Caffeine inhibited ACh-induced elevation of [Ca]i and secretin-induced fluctuation of [Ca]i. Caffeine abolished thapsigargin-induced intracellular Ca release but did not affect the entry of extracellular Ca. Caffeine (0.05 mM) abolished ethanol (1 mM)-induced fluid hypersecretion in secretin-stimulated pancreatic duct. Low concentrations of caffeine directly inhibit pancreatic ductal fluid secretion stimulated by secretin or ACh and also ethanol-induced fluid hypersecretion. The inhibition by caffeine seems to be mediated by the blockade of intracellular Ca mobilization. Daily intake of caffeine may reduce the volume of pancreatic juice secretion.

Quantitative determination and classification of energy drinks using near-infrared spectroscopy.

Almost a hundred commercially available energy drink samples from Hungary, Slovakia, and Greece were collected for the quantitative determination of their caffeine and sugar content with FT-NIR spectroscopy and high-performance liquid chromatography (HPLC). Calibration models were built with partial least-squares regression (PLSR). An HPLC-UV method was used to measure the reference values for caffeine content, while sugar contents were measured with the Schoorl method. Both the nominal sugar content (as indicated on the cans) and the measured sugar concentration were used as references. Although the Schoorl method has larger error and bias, appropriate models could be developed using both references. The validation of the models was based on sevenfold cross-validation and external validation. FT-NIR analysis is a good candidate to replace the HPLC-UV method, because it is much cheaper than any chromatographic method, while it is also more time-efficient. The combination of FT-NIR with multidimensional chemometric techniques like PLSR can be a good option for the detection of low caffeine concentrations in energy drinks. Moreover, three types of energy drinks that contain (i) taurine, (ii) arginine, and (iii) none of these two components were classified correctly using principal component analysis and linear discriminant analysis. Such classifications are important for the detection of adulterated samples and for quality control, as well. In this case, more than a hundred samples were used for the evaluation. The classification was validated with cross-validation and several randomization tests (X-scrambling). Graphical Abstract The way of energy drinks from cans to appropriate chemometric models.

Efficacy of different caffeine concentrations on growth and ochratoxin A production by Aspergillus species.

The objective of this study was to evaluate the effect of different caffeine concentrations (0-4%) on (i) lag phase prior to growth, (ii) growth rates and (iii) ochratoxin A (OTA) production by strains from the Aspergillus section Circumdati and Aspergillus section Nigri groups, isolated from coffee, when grown on a conducive medium at 0·98 water activity and 30°C. The lag phases prior to growth increased with caffeine concentration. A strain of Aspergillus niger and Aspergillus carbonarius were the most sensitive to caffeine with growth being inhibited by <1% caffeine. For strains of Aspergillus westerdijkiae, Aspergillus ochraceus and Aspergillus steynii, although growth was inhibited significantly, some growth (10-15% of controls) occurred in 4% caffeine. OTA production was significantly inhibited by only 0·5% caffeine for strains of A.westerdijkiae, A.niger and A.carbonarius. For A.steynii at least 1·5% caffeine was required to inhibit OTA production. In contrast, for the strain of A.ochraceus there was a stimulation of OTA at 3% with a reduction at 4% caffeine. These results are discussed in the context of the different concentrations of caffeine found in Arabica and Robusta coffee and the development of minimization strategies. Arabic (0·6%) and Robusta coffee (4%) have significantly different amounts of endogenous caffeine. The growth of six ochratoxigenic fungi which contaminate coffee with ochratoxin A (OTA) had differential tolerance/sensitivity to concentrations of caffeine invitro in this range. However, low concentrations of caffeine (<0·5%) was inhibitory to OTA production. These results are discussed in the context of the potential for using such information for the design of minimization strategies to control mycotoxin production in such products.

Low concentrations of caffeine induce asymmetric cell division as observed in vitro by means of the CBMN-assay and iFISH

The dual role of caffeine as a chromosomal damage inducer and G2/M-checkpoint abrogator is well known but it is observed mainly at relatively high concentrations. At low concentrations, caffeine enhances the cytogenetic effects of several carcinogens and its intake during pregnancy has been recently reported to cause adverse birth outcomes. Interestingly, a threshold below which this association is not apparent was not identified. Since chromosomal abnormalities and aneuploidy are the major genetic etiologies of spontaneous abortions and adverse birth outcomes, we re-evaluate here the effects of caffeine at the cytogenetic level and propose a model for the mechanisms involved. Our hypothesis is that low caffeine concentrations affect DNA replication and cause chromosomal aberrations and asymmetric cell divisions not easily detected at metaphase since damaged cells are delayed during their G2/M-phase transition and the low caffeine concentrations cannot abrogate the G2-checkpoint. To test this hypothesis, caffeine-induced chromatid breaks and micronuclei in peripheral blood lymphocytes (PBLs) were evaluated in vitro after low caffeine concentration exposures, followed by a short treatment with 4mM of caffeine to abrogate the G2-checkpoint. The results show a statistically significant increase in chromatid breaks at caffeine concentrations ≥1mM. When caffeine was applied for G2/M-checkpoint abrogation, a statistically significant increase in chromatid breaks, compared to an active checkpoint, was only observed at 4mM of caffeine. The potential of low concentrations to induce asymmetric cell divisions was tested by applying a methodology combining the cytochalasin-B mediated cytokinesis-block micronucleus assay (CBMN) with interphase FISH (iFISH), using selected centromeric probes. Interestingly, low caffeine concentrations induce a dose dependent aneuploidy through asymmetric cell divisions, which are caused by misalignment of chromosomes through a mechanism unrelated to the formation of chromatid breaks. The cytogenetic approach used, combining CBMN with iFISH, is proposed as a valuable tool to test chemically induced asymmetric cell divisions.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA.

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.

Effect of Purine Alkaloids on the Proliferation of Lettuce Cells Derived from Protoplasts

To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 μM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with > 250 μM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

Ryanodine receptor sensitization results in abnormal calcium signaling in airway smooth muscle cells.

Intracellular Ca(2+) dynamics of airway smooth muscle cells (ASMCs) are believed to play a major role in airway hyperresponsiveness and remodeling in asthma. Prior studies have underscored a prominent role for inositol 1,4,5-triphosphate (IP3) receptors in normal agonist-induced Ca(2+) oscillations, whereas ryanodine receptors (RyRs) appear to remain closed during such Ca(2+) oscillations, which mediate ASMC contraction. Nevertheless, RyRs have been hypothesized to play a role in hyperresponsive Ca(2+) signaling. This could be explained by RyRs being "sensitized" to open more frequently by certain compounds. We investigate the implications of RyR sensitization on Ca(2+) dynamics in ASMC using a combination of mathematical modeling and experiments with mouse precision-cut lung slices. Caffeine is used to increase the sensitivity of RyRs to cytosolic Ca(2+) concentration ([Ca(2+)]i) and sarcoplasmic reticulum Ca(2+) ([Ca(2+)]SR). In ASMCs, high caffeine concentrations (>10 mM) induce a sustained elevation of [Ca(2+)]i. Our mathematical model accounts for this by the activation of store-operated Ca(2+) entry that results from a large increase in the RyR sensitivity to [Ca(2+)]SR and the associated Ca(2+) release, which leads to a reduction of [Ca(2+)]SR. Importantly, our model also predicts that: (1) moderate RyR sensitization induces slow Ca(2+) oscillations, a result experimentally confirmed with low concentrations of caffeine; and (2) high RyR sensitization suppresses fast, agonist-induced Ca(2+) oscillations by inducing substantial store-operated Ca(2+) entry and elevated [Ca(2+)]i. These results suggest that RyR sensitization could play a role in ASMC proliferation (by inducing slow Ca(2+) oscillations) and in airway hyperresponsiveness (by inducing greater mean [Ca(2+)]i for similar levels of contractile agonist).

Caffeine increases sugar-sweetened beverage consumption in a free-living population: a randomised controlled trial.

Excessive sugar-sweetened beverage (SSB) consumption has been associated with overweight and obesity. Caffeine is a common additive to SSB, and through dependence effects, it has the potential to promote the consumption of caffeine-containing foods. The objective of the present study was to assess the influence that caffeine has on the consumption of SSB. Participants (n 99) were blindly assigned to either a caffeinated SSB (C-SSB) or a non-caffeinated SSB (NC-SSB) group. Following randomisation, all participants completed a 9 d flavour-conditioning paradigm. They then completed a 28 d ad libitum intake intervention where they consumed as much or as little of C-SSB or NC-SSB as desired. The amount consumed (ml) was recorded daily, 4 d diet diaries were collected and liking of SSB was assessed at the start and end of the intervention. Participants (n 50) consuming the C-SSB had a daily SSB intake of 419 (sd 298) ml (785 (sd 559) kJ/d) over the 28 d intervention, significantly more than participants (n 49) consuming the NC-SSB (273 (sd 278) ml/d, 512 (sd 521) kJ/d) (P=0.05). However, participants who consumed the C-SSB liked the SSB more than those who consumed the NC-SSB (6.3 v. 6.0 on a nine-point hedonic scale, P= 0.022). The addition of low concentrations of caffeine to the SSB significantly increases the consumption of the SSB. Regulating caffeine as a food additive may be an effective strategy to decrease the consumption of nutrient-poor high-energy foods and beverages.